EP2998406A1 - Méthode de suivi de métastase de cellules cancéreuses à l'aide de cellules cultivées dans un environnement tridimensionnel de collagène - Google Patents

Méthode de suivi de métastase de cellules cancéreuses à l'aide de cellules cultivées dans un environnement tridimensionnel de collagène Download PDF

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EP2998406A1
EP2998406A1 EP14797327.5A EP14797327A EP2998406A1 EP 2998406 A1 EP2998406 A1 EP 2998406A1 EP 14797327 A EP14797327 A EP 14797327A EP 2998406 A1 EP2998406 A1 EP 2998406A1
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metastasis
expression
cancer
cortactin
invasion
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Jung Weon Lee
Sunghoon Kim
Mi-Sook Lee
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Medicinal Bioconvergence Research Center
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Medicinal Bioconvergence Research Center
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Definitions

  • the present invention relates to a method for monitoring migration, invasion, and metastasis of cancer cells by observing the shape of cancer cells cultured in a three-dimensional environment and measuring the activity, expression, and changes in the expression sites of proteins associated with metastasis, and the degradation of an extracellular matrix; and to a method for screening a cancer metastasis inhibitor.
  • Metastasis is the process of the dissemination of cells from the primary tumor, by which cancer cells can be spread wide.
  • the said primary tumor can be developed by various genetic reasons of a host, which makes the treatment of each individual difficult.
  • metastasis is the general phenomenon observed in every cancer, so that it is an important target of therapeutic intervention.
  • the metastatic cancer cells leave the primary tumor, pass through basement membrane, and then invade into other tissues or organs.
  • the basement membrane is the supporting layer under epithelium that plays a role as an extracellular matrix (ECM) protein network. Once cancer cells are disseminated from the primary tumor, they travel through blood stream.
  • ECM extracellular matrix
  • the metastatic cancer cells form the invasive protrusions like invadopodia that is the suitable structure for cancer cells to invade blood vessels or lymphatic ducts with decomposing ECM and penetrating stromal layer.
  • Invadopodia the invasive protrusion wherein F-actin is accumulated, is located on the contact area of cells and matrix and has the capability of matrix degradation. This is the kind of structure where the intracellular signaling factors, protein degradation, cell adhesion, cytoskeleton, and membrane-trafficking pathway are gathered.
  • 3D cell culture can overcome the limit of the conventional cell culture and can make up the weakness of the animal model.
  • 3D cell culture method can provide an artificial control system by including or excluding a specific intracellular or microenvironmental factor therein, and is useful for verifying various hypotheses because the cell shape and signaling activity are closer to those of in vivo, and is advantageous in performing different experiments at the same time. It is also easy with 3D cell culture to observe the three-dimensional cell shape in a specific environment under microscope in real-time.
  • the basement membrane is a thin layer of specific ECM, which supports the epidermal layer and the endothelial layer and is composed of such proteins that connect cell and matrix as laminin, collagen, fibronectin, and entactin. These proteins play an important role in the regulatory mechanisms of cellular behavior including cell migration, adhesion, wound-healing, and scattering, etc.
  • a three-dimensional scaffold can be constructed in a lab with ECM. The three-dimensional scaffold plays a role as a temporary support for cells in a specific environment and then is eventually embodied in vivo. Scaffold has been widely used in the field of tissue engineering. Unlike the two-dimensional cell monolayer, this scaffold is a three-dimensional support having the original cell geometry.
  • the natural hydrogel such as type 1 collagen, type IV collagen, laminin, fibronectin, or hyaluronic acid.
  • the said natural hydrogel is physically weak but can provide a biological environment to cells.
  • the collagen hydrogel scaffold is often used for the construction of a three-dimensional organotypic breast cancer cell model. However, this collagen hydrogel cannot copy the real stiffness of the real tissue cells and cancer cells. In a three-dimensional culture, cells display various shapes according to the type, concentration, and hardness of the extracellular matrix (see Figure 13 ).
  • DCIS Ductal carcinoma in situ
  • IDC Invasive ductal carcinoma
  • IDC intracranial pressure
  • Invadopodia are the actin-rich protrusions observed on the cell membrane. Invadopodia are synthesized from the synthesis of actin core structure and can degrade the extracellular matrix by the accumulation of matrix metalloproteinase. Invadopodia have the function of metastasis and are mostly found in metastatic cancer cells. Invadopodia have a very similar shape to podosome in normal cells such as macrophages, monocytes, and osteoblasts where they can pass through the tissue barrier. In invadopodia (or invadosome), cortactin, tyrosine kinase, and such matrix metalloproteinases MT1-MMP are consolidated and coexist with actin. Unlike in a two-dimensional environment, the cells in a three-dimensional environment can produce invadopodia by changing the shape, cytoskeleton and contacts with matrix (see Figure 14 ).
  • JNK [c-Jun N-terminal kinase] is one of MAP (mitogen-activated protein) kinases and is activated by various steps and stimuli. In cancer cells, JNK induces apoptosis or increases cell survival and proliferation, indicating it is involved in both sides of cancer development. For example, the inhibition of JNK activity in some cancer cases could suppress the proliferation of cells or induce apoptosis. JNK activity and c-Jun phosphorylation are also necessary in the transformation induced by ras, the carcinogenic protein.
  • Snail1 is one of transcription factors which can be up-regulated in relation to epithelial mesenchymal transition (EMT). Snail1 binds to E-box element in E-cadherin gene promoter region and as a result it inhibits transcription and cell-cell adhesion, leading to EMT. Snail1 is induced by TGF ⁇ in various cell lines and regulates the expression of EMT related proteins, and regulates various cell functions including proliferation and apoptosis. The expression of snail1 is increased by TGF ⁇ signal activated by collagen in PDAC (pancreatic ductal adenocarcinoma).
  • PDAC pancreatic ductal adenocarcinoma
  • Snail1 regulates the process of extracellular fibrosis, but collagen is generated during the process and the produced collagen increases snail1 expression again, resulting in the increase of fibrosis. Snail1 and twist are accumulated in the leading edge of the growing mammary buds, and are accordingly involved in mammary epithelial branching.
  • the present inventors tried to develop a novel method for monitoring metastasis of cancer cells cultured in a three-dimensional extracellular matrix environment.
  • the inventors confirmed the decrease of c-Jun phosphorylation by JNK activity inhibition, the activation of smad proteins relating to TGF ⁇ 1 signaling, the increase of snail1 expression, and the decrease of cortactin expression when the breast cancer cell line MDA-MB-231 was cultured in a three-dimensional collagen gel environment or extracellular acidity was raised or hypoxia was induced by reducing the intracellular oxygen concentration.
  • the present inventors additionally confirmed that such interactions between molecules were identical to those in breast cancer tissues obtained from patients.
  • MT1-MMP could be used as another invadopodia marker in a three-dimensional collagen gel environment and the inhibition of JNK could increase snail1 expression.
  • the decrease of cortactin expression by snail1 had a negative effect on the location and role of MT1-MMP, resulting in the inhibition of invadopodia formation and the inhibition of the degradation of collagen matrix surrounding invadopodia.
  • the present invention provides a method for monitoring cancer cell migration, invasion, metastasis, and the degree of metastasis, comprising the following step:
  • the present invention also provides a method for screening a cancer metastasis inhibitor comprising the following steps:
  • the present invention can be used as a method for monitoring the effect of extracellular microenvironment on various cell functions by regulating the extracellular microenvironment in a three-dimensional culture that can copy in vivo environment, a method for monitoring cancer cell migration, invasion, metastasis, and the degree of metastasis by imaging the invadopodia formation in cancer cells cultured in a three-dimensional collagen gel environment, and a method for screening a cancer metastasis inhibitor.
  • the present invention can also be useful as one of screening methods capable of creating low-cost, high-efficient added value at the time of pre-clinical tests required for drug development.
  • invadopodia indicates the region wherein actin and cortactin can be expressed at the same time. Actin is polymerized and strengthened in the protrusion of cell membrane by the action of cortactin, where the matrix metalloprotease is accumulated to degrade extracellular matrix (ECM).
  • ECM extracellular matrix
  • invadopodia various proteins such as cortactin, gelsolin, vinculin, talin, and paxillin are gathered together, so that various signaling activities are happening there for actin-reconstruction so as to allow cancer cells to degrade matrix.
  • the present invention provides a method for monitoring cancer cell migration, invasion, metastasis, and the degree of metastasis, comprising the following step:
  • the cancer is preferably a metastatic cancer or a metastasis inducible cancer, which is preferably selected from the group consisting of breast cancer, liver cancer, stomach cancer, colon cancer, bone cancer, pancreatic cancer, head/neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small bowel neoplasm, anal cancer, colon carcinoma, fallopian tube carcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin's disease, prostatic cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic cancer, and central nervous system tumor.
  • the cancer is preferably breast cancer, but not always limited thereto.
  • the cell culture in step 1) is preferably performed under the regulation of cell culture period, cell number (density), extracellular pH, or extracellular oxygen level, but not always limited thereto.
  • the culture vessel of step 1) is preferably made of one of those materials selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins, polyimides, and polyurethanes, and polydimethylsiloxane was preferably selected in a preferred embodiment of the invention, but not always limited thereto.
  • PDMS polydimethylsiloxane
  • PMMA polymethylmethacrylate
  • polyacrylates polycarbonates
  • polycyclic olefins polyimides
  • polyurethanes polydimethylsiloxane was preferably selected in a preferred embodiment of the invention, but not always limited thereto.
  • a natural hydrogel well known to those in the art which is exemplified by collagen, laminin, fibronectin, or hyaluronic acid, can be used.
  • the collagen is preferably type 1 collagen, but not always limited thereto.
  • the concentration of the said type I collagen is preferably 1 ⁇ 5 mg/ml, more preferably 2 ⁇ 4 mg/ml, and most preferably 2.5 ⁇ 3 mg/ml.
  • the collagen is preferably prepared as a neutral, but not always limited thereto.
  • the cell culture in step 1) is performed with 1 x 10 4 ⁇ 2 x 10 8 cells/ml in the culture medium containing the said natural material, and more preferably with 1 x 10 5 ⁇ 2 x 10 7 cells/ml, and most preferably with 1 x 10 6 ⁇ 2 x 10 6 cells/ml, but not always limited thereto.
  • the change in the shape of the cells in step 2) is characterized by being longer and the contact region of cell and extracellular matrix (ECM) preferably becomes simply flat and the formation of invadopodia is preferably confirmed therein, but not always limited thereto.
  • ECM extracellular matrix
  • the expression site of the metastasis associated protein in step 2) is preferably changed from cell membrane to cytoplasm or around the nucleus, but not always limited thereto, and the changes of the location into other regions except cell membrane are also included.
  • the change in the activity of the metastasis associated protein in step 2) is preferably characterized by the increase of c-Jun phosphorylation, and the change of the expression is preferably characterized by the decrease of cortactin and the increase of snail1, but not always limited thereto.
  • the target protein for the investigation of changes in the activity and expression includes all of those proteins that are located in invadopodia and play an important role in the functions of invadopodia.
  • the cell migration and invasion in step 2) are preferably confirmed by measuring the degradation of collagen gel matrix, and more precisely by investigating the changes of collagen gel matrix degrading activity, but not always limited thereto.
  • the measurement of the activity and expression of the protein in step 2) is preferably performed by the method selected from the group consisting of Western blotting, RT-PCR, real-time PCR, immunofluorescence, ChIP (chromatin immunoprecipitation), EMSA (Electrophoric Mobility Shift Assay), or ECM degrading activity assay using DQTM-collagen type I, in a preferred embodiment of the invention, but not always limited thereto.
  • the present invention also provides a method for screening a cancer metastasis inhibitor comprising the following steps:
  • the inventors confirmed the decrease of c-Jun phosphorylation, the decrease of cortactin protein expression, and the increase of snail1 protein expression in the breast cancer cell line MDA-MB-231 cultured in a three-dimensional collagen gel environment (see Figure 1A ).
  • the identical results were observed when the microenvironment surrounding the cells was changed, for example cell density or extracellular acidity was changed or hypoxia was induced (see Figures 1B ⁇ 1D ).
  • the present inventors also investigated the effect of JNK inhibitor on the formation of invadopodia in various cell lines in a three-dimensional collagen gel culture environment.
  • JNK inhibition changed the cell shape, increased the snail1 expression, and reduced the cortactin expression (see Figures 4E , 4F , 4G and 4J ).
  • the numbers of spots where actin and cortactin coexist were reduced (see Figure 4I ), and the collagen matrix degrading activity was decreased (see Figure 4K ).
  • the present inventors also investigated the mechanism of the decrease of cortactin expression and the increase of snail1 expression by the treatment of JNK inhibitor in the breast cancer cell line cultured in a three-dimensional collagen gel environment, and further the inventors confirmed that JNK inhibition increased snail1 and the increased snail1 was conjugated to cortactin promoter to suppress cortactin expression (see Figures 6A ⁇ 6D, and 6H ). It was also confirmed that the regulation of cortactin mRNA or protein level by JNK inhibitor was controlled in the stage of transcription (see Figures 6E and 6G ).
  • the present invention also investigated the mechanism of the increase of snail1 mRNA expression according to the treatment of JNK inhibitor in the breast cancer cell line cultured in a three-dimensional collagen gel environment. As a result, it was confirmed that JNK inhibition caused the increase of TGF ⁇ and accordingly smad2 was up-regulated, and at the same time the phosphorylation of smad2 was also increased suggesting that snail1 was up-regulated (see Figures 7A ⁇ 7E ).
  • the present inventors further confirmed that when JNK inhibitor was treated to the breast cancer cell line cultured in a three-dimensional collagen gel environment, the up-regulated or activated smad protein was directly conjugated to snail1 promoter region to increase snail1 transcription (see Figure 7 F) .
  • JNK1 siRNA was treated to the cells, the result was consistent with those shown in the above Figure 8A and Figure 8B (see Figures 8C ⁇ 8E ).
  • the inventors also confirmed that the expression of cortactin was decreased when snail1 was over-expressed in the breast cancer cell line cultured in a three-dimensional collagen gel environment (see Figures 9A ⁇ 9C ).
  • snail1 was knocked-down after JNK inhibition, the JNK inhibitor dependent snail1 expression was not induced anymore and the expression of cortactin was not decreased any further (see Figures 9D and 9E ). Therefore, it was confirmed that snail1 expression played an important role in cortactin expression and had a negative effect on the formation of invadopodia.
  • the present inventors confirmed that the degradation of collagen was observed (yellow arrow) in the breast cancer cell line cultured in a three-dimensional collagen gel environment containing green fluorescence dye-conjugated collagen, but the degradation was not observed in the cells treated with JNK inhibitor (see Figure 10A ).
  • the inventors also confirmed that MT1-MMP protein could be used as an invadopodia marker in addition to cortactin (see Figure 10B ).
  • the expression and the expression site of MT1-MMP were changed dynamically on the cell membrane starting from the edge of the cell membrane toward the moving direction of the cell (see Figure 10C ).
  • JNK inhibitor when treated to the breast cancer cell line cultured in a three-dimensional collagen gel environment, changed the position of the co-expression of cortactin and MT1-MMP from the cell membrane to near the nucleus in the cytoplasm, and as a result cell invasion associated functions including ECM degradation could not be normally functioning (see Figure 11 ).
  • JNK inhibitor when treated to the breast cancer cell line cultured in a three-dimensional collagen gel environment, suppressed MT1-MMP functions and thereby the DQ-collagen and type I collagen matrix degrading activity was decreased (see Figure 12 ).
  • JNK inhibition in MDA-MB-231 cell line cultured in a three-dimensional collagen gel environment caused the increase of snail1 expression but the decrease of cortactin expression, and at the same time had a negative effect on the location and role of cortactin and MT1-MMP, and as a result the formation of invadopodia and the degradation of type I collagen matrix were suppressed.
  • Example 1 Changes of intracellular protein caused by microenvironment in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • the PDMS culture vessel equipped with a cover glass on one side was prepared.
  • PDMS crude liquid was mixed with a hardener at the ratio of 10:1, which was hardened at 100°C for 1 hour.
  • the hardened PDMS was taken off from the mold and punched by using an 8 mm punch.
  • a cover glass 24 x 60 mm, Marienfeld
  • the prepared PDMS was used after being irradiated with UV.
  • MDA-MB-231, MDA-MB-436, MDA-MB-468, T47D, BT549, Hs578T, and MCF7 (ATCC, USA) cell lines were cultured by using PureCol type I collagen (bovine collagen I; Advanced BioMatrix, USA) or matrigel (BD Bioscience, USA). At this time, the final concentration of collagen was 2.5 ⁇ 3 mg/ml and the concentration of matrigel was 4 ⁇ 10 mg/ml.
  • the strong acid collagen (pH 2) solution was adjusted to be neutral (pH 7) by using 10x reconstitution buffer [260 mM sodium bicarbonate, 250 mM HEPES, 2 N NaOH, and serum-free 10x RPMI (Sigma, USA)] so as not to induce any changes in the cell.
  • the prepared collagen solution was stored at 4°C for 10 minutes until the collagen fibers were fully formed.
  • 10 ⁇ l of the solution was poured in the PDMS vessel of 8 mm in the diameter, which stood in a 37 °C incubator for 30 minutes.
  • MDA-MB-231 cell line which was cultured in PRMI-1640 or DMEM (JBI, Korea) supplemented with 10% FBS and penicillin/streptomycin (Invitrogen, USA), was washed with PBS twice. Then, the cells were taken off by using trypsin. The collected cells were precipitated by centrifugation, and the numbers of the cells were counted by using a hematocytometer. The cells (1 ⁇ 2 x 10 6 cells/ml) were well-mixed in the prepared collagen solution, which was loaded in the PDMS culture vessel covered with collagen or matrigel. The culture vessel was placed in a 37 °C incubator for 30 minutes ⁇ 1 hour to harden the collagen or matrigel, followed by culture.
  • the collagen gel which was mixed with the MDA-MB-231 cell line cultured in the PDMS culture vessel by the method of Example ⁇ 1-1> was collected in a microcentrifuge-tube, followed by centrifugation at 5000 rpm for 1 minute.
  • collagen gel and cell pellet were washed with cold PBS (130 mM NaCl, 13 mM Na 2 HPO 4 , 3.5 mM NaH 2 PO 4 , pH 7.4) twice, to which certain amount of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 0.25% sodium deoxycholate) supplemented with protease inhibitor cocktails (GenDepot) was added, followed by lysis 4°C for 1 hour. The lysed sample was centrifuged at 13000 rpm for 30 minutes.
  • PBS 130 mM NaCl, 13 mM Na 2 HPO 4 , 3.5 mM NaH 2 PO 4 , pH 7.4
  • certain amount of lysis buffer 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 0.25% sodium deoxycholate
  • protease inhibitor cocktails GenDepot
  • the obtained supernatant was added with 4x sample buffer [200 mM Tris-HCl (pH 6.8), 8% SDS, 0.4% bromophenol blue, 40% glycerol], followed by 10 ⁇ 12% SDS-PAGE. Then, the proteins were transferred onto Nitrocellulose Membranes ProtranTM nitrocellulose membrane (Whatman), followed by pre-treatment with 5% skim milk.
  • the membrane was washed with PBS (130 mM NaCl, 13 mM Na 2 HPO 4 , 3.5 mM NaH 2 PO 4 , pH 7.4) twice, followed by reaction at 4°C for 15 hours with the mouse monoclonal antibodies of anti-E-cadherin (24E10), smad2, smad3, phospho-smad2, phospho-smad3, phospho-MAPKAPK-2 (Thr222), phospho-Ser 63 -c-Jun, c-Jun, snail1 (L70G2) (Cell signaling, USA), cortactin, HIF1 alpha (BD bioscience, USA), slug, JNK, PCNA (Santa Cruz Biotechnology, USA), anti-MT1-MMP (Millipore, USA), and TGF ⁇ (1, 2, 3) (R&D systems, USA).
  • PBS 130 mM NaCl, 13 mM Na 2 HPO 4 , 3.5 mM NaH 2 PO 4 , pH 7.4
  • Example ⁇ 1-3> To investigate whether or not the results of Example ⁇ 1-3> could be consistent in the human breast cancer tissues, immunohistochemical staining was performed with the breast cancer patient tissues.
  • the tissues obtained from 2 breast cancer patients were respectively fixed in 4% paraformaldehyde.
  • the paraffin block was sliced in 4 ⁇ m thickness, and the thin sections were dried to obtain paraffin sections.
  • the paraffin-embedded tissue slide was deparaffinized and then rehydrated, followed by the treatment with 3% hydrogen peroxide for 10 minutes.
  • One section of the slide was placed in 10 mM citrate buffer (pH 6.0), which was boiled for 20 minutes.
  • the slide was reacted with the antibodies of pS63-c-Jun, snail1, and cortactin at 4°C for at least 18 hours. Immunohistochemical staining was performed by using streptavidin-conjugated peroxidase as the secondary antibody.
  • Example 2 Changes of cell shape and cell migration by JNK inhibitor in the breast cancer cell line cultured in a three-dimensional collagen gel environment ⁇ 2-1> Changes of cell shape by JNK inhibitor
  • the culture medium supplemented with 10% FBS (control) and the culture medium supplemented with 50 ⁇ M of SP600125 (experimental group) were loaded on top of the gel, followed by culture for 3 days. Then, the shape of the cells and the migration pattern in the gel were observed under microscope.
  • RT-PCR and Western blotting were performed to investigate the changes in mRNA and protein expressions in the MDA-MB-231 cell line cultured in a three-dimensional collagen gel environment induced by the treatment of JNK inhibitor.
  • mRNA was first prepared from the total RNA obtained from the cell line treated with SP600125 as shown in Example ⁇ 2-1> by using TRIzol® (Invitrogen, USA). Then, cDNA was synthesized by using AmfiRivert cDNA Synthesis Master Mix (GenDePot). PCR was performed with Thermo Scientific DreamTaq Green PCR Master Mix (Thermo Scientific). The primers used for PCR were as shown in Table 1. After PCR, electrophoresis was performed to confirm the bands on agarose gel. The band intensities were measured by using Image J. The expression level was modified by total mRNA, considering GAPDH mRNA as a standard, and then relative ratios were calculated.
  • cortactin protein was over-expressed in the cells treated with JNK inhibitor. Then, the shape of the cells was observed.
  • cortactin was induced in the cell line treated with SP600125 and cultured in Example ⁇ 2-1>, followed by further culture in a three-dimensional collagen gel environment for 3 days.
  • the shape of the cells and the migration in the gel were observed under microscope.
  • time-lapse imaging was performed to screen the changes in cell shape and cell migration in real-time.
  • the culture medium supplemented with 10% FBS (control) and the culture medium supplemented with 50 ⁇ M of SP600125 were loaded on top of the gel, followed by culture for 3 days.
  • the culture medium was replaced every other day.
  • the control group hardened in an incubator for 30 minutes ⁇ 1 hour for time-lapse imaging and the experimental group treated with SP600125 proceeded to imaging with Olympus IX81-ZDC microscope, wherein images were obtained every 30 minutes, photo by photo, for 20 hours at 37 °C in the presence of 5% CO 2 .
  • Example 2 it was investigated whether or not the above results obtained in Example 2 were consistent with those resulted from the culture in a matrigel (another ECM) environment.
  • MDA-MB-231 cell line was cultured in a three-dimensional matrigel environment treated with SP600125 and then the shape of the cells was investigated by the method of Example ⁇ 2-1>.
  • the changes in protein expression were also investigated by the method of Example ⁇ 1-3>.
  • Example 4 Reduction of cortactin expression by JNK inhibitor in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • the MDA-MB-231 cell line cultured in a three-dimensional collagen gel environment and treated with SP600125 was fixed in 4% formaldehyde (Sigma, USA) for 30 minutes. Then, the formaldehyde was eliminated and reaction was induced in 100 mM PBS glycine solution for 30 minutes, followed by permeabilization using 0.5% triton X-100 for 30 minutes. The reaction time could be adjusted in order for the cells in the collagen gel to be fully contacted with the solution. Then, the cells were pre-treated with 3% BSA solution for 2 hours. F-actin was stained with rhodamine palloidine (red) at room temperature for at least 4 hours.
  • the cells were washed with washing buffer [0.2% triton X-100, 0.1% BSA, and 0.05% Tween 20 were added to PBS solution (pH 7.4), which was sterilized by using 0.22 ⁇ m filter], and stained with Alexa488 ® -conjugated cortactin (green) antibody at 4°C for at least 18 hours. Lastly, DAPI (4',6-diamidino-2-phenylindole; blue, Molecular Probe) staining was performed to observe the shape of nucleus. The antibody reaction time was adjusted according to the intensity of staining. The stained cells were observed under Olympus FV1000 confocal microscope and Nikon Eclipse Ti confocal microscope. Z-stack images obtained from the confocal microscope were reconstructed as 3D images by using Easy 3D modes of IMARIS software. The co-localization of the reconstructed images was analyzed by using ImarisColoc and surpass module, followed by visualization.
  • MDA-MB-436, MDA-MB-468, MDA-MB-453, T47D, BT549, Hs578T, and MCF7 cell lines were cultured in a three-dimensional collagen gel environment treated with SP600125.
  • Immunofluorescence staining was performed by the same manner as described in Example ⁇ 4-1> and as a result the formation of invadopodia was confirmed.
  • Western blotting was also performed by the same manner as described in Example ⁇ 1-3> to measure the expression levels of intracellular proteins.
  • Example 5 Mechanism of the increase of snail1 expression and the decrease of cortactin expression by JNK inhibitor in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • ChIP chromatin immunoprecipitation
  • the MDA-MB-231 cell line cultured in a three-dimensional collagen gel environment was fixed in 4% formaldehyde for 30 minutes.
  • the cells were then treated with 1.5 M glycine, followed by washing with cold PBS containing protease inhibitor.
  • the fixed cells were loaded in SDS-lysis buffer supplemented with protease/kinase inhibitor, followed by sonication for lysis.
  • the lysed sample proceeded to electrophoresis at 13000 rpm for 3 minutes, and the supernatant was transferred into a microcentrifuge tube, followed by sonication again for chromatin fragmentation.
  • TE solution was added thereto, followed by reaction at 65°C for 15 hours.
  • RNase A was added to each input and sample, followed by reaction at 37°C.
  • Proteinase K was added thereto, followed by reaction at 55°C for 1 hour.
  • Phenol/chloroform (1:1) was added thereto, followed by vortexing and then centrifugation was performed.
  • supernatant was collected, to which TE/200 mM NaCl and glycogen were added, followed by centrifugation, leading to the ethanol precipitation.
  • the DNA pellet obtained from the centrifugation was naturally dried and then dissolved in sterilized distilled water. PCR was performed with the primers listed in Table 2.
  • snail1 is conjugated to E-cadherin promoter region to inhibit E-cadherin expression and snail1 is recognized as a transcription inhibitor that suppresses gene transcription ( Hemavathy K, Ashraf SI, Ip YT. Snail/slug family of repressors: slowly going into the fast lane of development and cancer. Gene. 2000;257:1-12 .). Based on that, the present inventors confirmed once again that the snail1 increased by JNK inhibition was conjugated to cortactin promoter region and accordingly the expression of cortactin was suppressed.
  • Example ⁇ 5-1> which is the formation of snail1 and cortactin promoter protein-DNA complex
  • EMSA electrospray mobility shift assay
  • a nuclear extract was prepared from the MDA-MB-231 cell pellet cultured in a three-dimensional collagen gel environment by using buffer C containing 50 mM HEPES pH 7.9, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 10% glycerol. Then, 10 ⁇ 15 ⁇ g of the nuclear extract was reacted with 2 nM of 32 P-labeled probe (SEQ. ID.
  • reaction mixture was expanded on nondenaturing polyacrylamide gel at 70 V for 2 hours by using 0.5 ⁇ TBE (Tris/borate/EDTA, pH 8) buffer containing 90 mM Tris base, 90 mM borate, and 0.5 mM EDTA, followed by drying for 2 hours.
  • the isotope was exposed on X-ray film to confirm snail1-DNA complex.
  • snail1 antibody Cell Signaling Technology, Inc.
  • snail1 antibody Cell Signaling Technology, Inc.
  • Example ⁇ 5-1> could be regulated at mRNA level or at protein level.
  • TGF ⁇ 1 pathway is induced by the conjugation between the ligand TGF ⁇ 1 and its receptors TGF ⁇ 1-receptor I and TGF ⁇ 1-receptor II.
  • TGF ⁇ 1 receptors When these receptors are activated by TGF ⁇ 1, the phosphorylation and activation of smad2 or smad3 are induced, leading to the formation of a complex with smad4. Then, the complex moves into nucleus and acts as a transcription factor therein. Therefore, the present inventors measured the levels of cortactin, smad2, smad3, and snail1 mRNAs in the cells cultured for 5 days by the same manner as described in Example ⁇ 2-2>. The protein expression level was also examined by the same manner as described in Example ⁇ 1-3>.
  • JNK inhibition in a three-dimensional collagen gel environment caused the increase of TGF ⁇ 1 and accordingly caused the increase of smad2 and at the same time the increase of smad2 phosphorylation together with the increase of snail1 expression.
  • smad has the binding element so called "CAGA" in a three-dimensional collagen gel environment (Dennler et al., 1998). So, it was first examined that such smad binding element was there in snail1 promoter region. Then, primers were designed with the region presumed where the smad binding element was located in snail1 promoter region, followed by ChIP using the primers listed in Table 2.
  • Example 6 Reduction of invadopodia formation by the inhibition of JNK activity and the decrease of JNK protein level in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • Example 7 Changes in cell shape according to the increase of snail1 expression in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • Example 4 Western blotting was performed by the same manner as described in Example ⁇ 1-3> and immunofluorescence staining was performed by the same manner as described in Example 4 in order to investigate the regulation of cortactin expression, cell shape, and actin-enriched spot population according to the regulation of snail1 expression.
  • Example 8 Confirmation of invadopodia marker in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • Example 9 Mechanism of cell migration by the regulation of MT1-MMP expression site by JNK activation in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • MDA-MB-231 cells were transfected with mCherry expression vector harboring the labeled MT1-MMP cDNA for 48 hours. Then, the cells were mixed with 2.5 mg/ml of type I collagen solution at the density of 10 6 cells/ml. 70 ⁇ l, of the cell/collagen mixture was loaded in PDMS vessel, followed by solidification at 37 °C for 1 hour. As for the experimental group, 50 ⁇ M of SP600125, the JNK inhibitor, was treated to the medium. As for the control, the medium containing 10% FBS not treated with INK inhibitor was treated. Both were cultured for 24 hours.
  • Any changes in dynamism of MT1-MMP expression site were traced by observing MT1-MMP location under Nikon T1 confocal microscope in real-time. At this time, to track down the location of MT1-MMP, 5 sites were selected per each sample, followed by imaging for 4 hours with taking photographs of 7 z-stacks every 5 minutes.
  • Example 10 Changes in the cell migration pattern by the co-expression of cortactin and MT1-MMP in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • JNK inhibition caused the reduction of cortactin expression in the MDA-MB-231 cultured in a three-dimensional collagen gel environment and thereby directly affected cell migration and invasion, but did not affect MT1-MMP expression.
  • GFP-cortactin and mCherry-MT1-MMP were co-expressed, followed by treating JNK inhibitor.
  • each protein was examined to see how each protein was affected by the treatment of JNK inhibitor, by the same manner as described in Example 9.
  • JNK inhibition in the MDA-MB-231 cell line cultured in a three-dimensional collagen gel environment caused the increase of snail1 expression via TGF ⁇ 1/smad expression and signaling activity, and accordingly caused the decrease of cortactin expression, and at the same time inhibited the formation of invadopodia by negatively affecting the location and role of MT1-MMP, resulting in the inhibition of cell invasion.
  • Example 11 Inhibition of type I collagen matrix degradation and suppression of MT1-MMP functions by JNK inhibitor in the breast cancer cell line cultured in a three-dimensional collagen gel environment
  • MDA-MB-231 cells were transfected with mCherry-labeled MT1-MMP or the control vector gene, followed by culture for 48 hours.
  • 2.5 mg/ml of 3D collagen type I (PureCol) and 2.5 mg/ml of DQTM-collagen I (Life Technologies) were mixed at the ratio of 10:1 (w:w), resulting in the preparation of collagen gel, in which the cultured cells were embedded at the density of 1.5 ⁇ 10 6 cells/ml.
  • the gel was solidified at 37°C for 30 minutes, on which 10% FBS/RPMI-1640 medium supplemented with 200 ml of DMSO or 50 mM SP600125 was added, followed by further culture.

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